1. Field of the Invention
The present invention relates to novel antiproliferative genes. More specifically, isolated nucleic acid molecules are provided encoding the human B-cell translocation genes 2 and 3 (BTG-2 and BTG-3). BTG-2 and BTG-3 polypeptides are also provided, as are vectors, host cells and recombinant methods for producing the same.
2. Related Art
In normal tissues, homeostasis is maintained through negative and positive growth controls which effect the proliferation and differentiation related to cellular genetic programs. An alteration of this subtle balance can result in developmental abnormalities or in neoplasia. Proto-oncogenes, genes that promote cell division, were the first growth-inducing elements to be identified and more than sixty of them have been described so far (Bishop, J. M., Cell 64:235:248 (1991)). The genes that negatively regulate cell proliferation are crucial to counteract the growth-inducing elements and are likely to have the same importance as proto-oncogenes in controlling cell division (Marshall, C. J., Cell 64:313-326 (1991)), especially since the loss of their function has been reported to be associated with irregular cellular differentiation and proliferation or with alteration of embryonic development (Weinberg, R. A., Science 254:1138-1146 (1991)).
The polynucleotides and polypeptides of the present invention are thought to be members of a family of anti-proliferative genes. BTG-1 is a member of this group and has been cloned and expressed. (Rovault, J. P. et al., The EMBO Journal 11(4):1663-1670 (1992)). BTG-1 was shown to negatively regulate N1H3T3 cell proliferation when over- or inappropriately expressed. BTG stands for B-cell translocation gene, and the BTG-1 gene has been shown to be involved in a chromosomal translocation [t(8;12)(q24;22)] in B-cell chronic lymphocytic leukemia.
The BTG-1 open reading frame is 60% homologous to PC3, an immediate early gene induced by nerve growth factor in rat PC12 cells. Sequence and Northern blot analyses indicate that BTG-1 and PC3 are not cognate genes but are thought to be members of this new family of anti-proliferation genes. The BTG-1 gene is preferentially expressed in quiescent cells during the early sub-phases of G1 in a serum-dependent manner and it is then down-regulated to reach a minimum level as the cells enter the S phase. This suggests a functional link between BTG-1 and the cell cycle process. BTG-1 is expressed in tissues (lymphoid, liver, placenta) containing non-dividing cells likely to re-enter the cell cycle upon different stimuli, whereas the expression of BTG-1 is barely detectable in fully differentiated tissues such as brain and muscle.
The BTG-1 gene was shown to be highly conserved in evolution and a similar 1.8 Kb transcript can be detected in murine and chicken tissue by using a human BTG-1 DNA probe (Rimokh, R. et al., Genes Chrom. Cancer 3:24-36 (1991)).
The BTG-2 and BTG-3 genes and gene products have been putatively identified as members of this family as a result of amino acid sequence homology to BTG-1.
The present invention provides isolated nucleic acid molecules comprising a polynucleotide encoding the BTG-2 or BTG-3 polypeptide having the amino acid sequences shown in FIGS. 1A-1B (SEQ ID NO:2) and FIGS. 2A-2B (SEQ ID NO:4), respectively, or having the amino acid sequences encoded by the cDNA clones deposited as ATCC Deposit Number 97025 on Jan. 17, 1995 (BTG-2) and ATCC Deposit Number 97010 on Jan. 5, 1995 (BTG-3). For BTG-2, the nucleotide sequence determined by sequencing the deposited cDNA clone contains an open reading frame encoding a polypeptide of about 345 amino acid residues, with a predicted leader sequence of about 25 amino acid residues such that the mature protein comprises about 320 amino acids. The amino acid sequence of the predicted mature BTG-2 protein is shown in FIGS. 1A-1B, amino acid residues from about 26 to about 345 (SEQ ID NO:2).
For BTG-3, the nucleotide sequence determined by sequencing the deposited cDNA clone contains an open reading frame encoding a polypeptide of about 344 amino acid residues, with a predicted leader sequence of about 18 amino acid residues such that the mature protein comprises about 326 amino acids. The amino acid sequence of the predicted mature BTG-3 protein is shown in FIGS. 2A-2B, amino acid residues from about 19 to about 344 (SEQ ID NO:4).
Thus, one aspect of the invention provides an isolated nucleic acid molecule comprising a polynucleotide having a nucleotide sequence selected from the group consisting of: (a) a nucleotide sequence encoding the BTG-2 or BTG-3 polypeptide having the complete amino acid sequence in FIGS. 1A-1B (SEQ ID NO:2) and FIGS. 2A-2B (SEQ ID NO:4), respectively; (b) a nucleotide sequence encoding the mature BTG-2 or mature BTG-3 polypeptide having the amino acid sequence at positions from about 26 to about 345 in FIGS. 1A-1B (SEQ ID NO:2) and from about 19 to about 344 in FIGS. 2A-2B (SEQ ID NO:4), respectively; (c) a nucleotide sequence encoding the BTG-2 or BTG-3 polypeptide having the complete amino acid sequence encoded by the eDNA clone contained in ATCC Deposit No. 97025 and 97010, respectively; (d) a nucleotide sequence encoding the mature BTG-2 or mature BTG-3 polypeptide having the amino acid sequence encoded by the eDNA clone contained in ATCC Deposit No. 97025 and 97010, respectively; and (e) a nucleotide sequence complementary to any of the nucleotide sequences in (a), (b), (c) or (d) above.
Further embodiments of the invention include isolated nucleic acid molecules that comprise a polynucleotide having a nucleotide sequence at least 90% identical, and more preferably at least 95%, 96%, 97%, 98% or 99% identical, to any of the nucleotide sequences in (a), (b), (c), (d) or (e), above, or a polynucleotide which hybridizes under stringent hybridization conditions to a polynucleotide in (a), (b), (c), (d) or (e), above. This polynucleotide which hybridizes does not hybridize under stringent hybridization conditions to a polynucleotide having a nucleotide sequence consisting of only A residues or of only T residues. An additional nucleic acid embodiment of the invention relates to an isolated nucleic acid molecule comprising a polynucleotide which encodes the amino acid sequence of an epitope-bearing portion of a BTG-2 or BTG-3 polypeptide having an amino acid sequence in (a), (b), (c) or (d), above.
The present invention also relates to recombinant vectors, which include the isolated nucleic acid molecules of the present invention, and to host cells containing the recombinant vectors, as well as to methods of making such vectors and host cells and for using them for production of BTG-2 or BTG-3 polypeptides or peptides by recombinant techniques.
The invention further provides an isolated BTG-2 or BTG-3 polypeptide having an amino acid sequence selected from the group consisting of: (a) the amino acid sequence of the BTG-2 or BTG-3 polypeptide having the complete 345 and 344 amino acid sequences shown in FIGS. 1A-1B (SEQ ID NO:2) and FIGS. 2A-2B (SEQ ID NO:4), respectively; (b) the amino acid sequence of the predicted mature BTG-2 or BTG-3 polypeptide (without the leader) having the amino acid sequence at positions from about 26 to about 345 in FIGS. 1A-1B (SEQ ID NO:2) and from about 19 to about 344 in FIGS. 2A-2B (SEQ ID NO:4), respectively; (c) the amino acid sequence of the BTG-2 or BTG-3 polypeptide having the complete amino acid sequence, including the leader, encoded by the cDNA clone contained in ATCC Deposit No. 97025 and 97010, respectively; and (d) the amino acid sequence of the mature BTG-2 or BTG-3 polypeptide having the amino acid sequence encoded by the cDNA clone contained in ATCC Deposit No. 97025 and 97010, respectively. The polypeptides of the present invention also include polypeptides having an amino acid sequence with at least 90% similarity, and more preferably at least 95% similarity to those described in (a), (b), (c) or (d) above, as well as polypeptides having an amino acid sequence at least 80% identical, more preferably at least 90% identical, and still more preferably 95%, 96%, 97%, 98% or 99% identical to those above.
An additional embodiment of this aspect of the invention relates to a peptide or polypeptide which has the amino acid sequence of an epitope-bearing portion of a BTG-2 or BTG-3 polypeptide having an amino acid sequence described in (a), (b), (c) or (d), above. Peptides or polypeptides having the amino acid sequence of an epitope-bearing portion of a BTG-2 or BTG-3 polypeptide of the invention include portions of such polypeptides with at least six or seven, preferably at least nine, and more preferably at least about 30 amino acids to about 50 amino acids, although epitope-bearing polypeptides of any length up to and including the entire amino acid sequence of a polypeptide of the invention described above also are included in the invention.
In another embodiment, the invention provides an isolated antibody that binds specifically to a BTG-2 or BTG-3 polypeptide having an amino acid sequence described in (a), (b), (c) or (d) above. Such antibodies are useful diagnostically or therapeutically as describe below.
In accordance with yet a further aspect of the present invention, there is provided a process for utilizing such polypeptides, or polynucleotides encoding such polypeptides for therapeutic purposes, for example, to treat disease states characterized by aberrant cellular proliferation, and to modulate cellular growth.
In accordance with yet another aspect of the present invention, there are provided antagonists to such polypeptides, which may be used to inhibit the action of such polypeptides, for example, in the treatment of diseases related to chromosomal translocation, for example, lymphocytic leukemia.
In accordance with still another aspect of the present invention, there are provided diagnostic assays for detecting diseases related to the under-expression of the polypeptides of the present invention and mutations in the nucleic acid sequences encoding such polypeptides.
In accordance with yet a further aspect of the present invention, there is provided a process for utilizing such polypeptides, or polynucleotides encoding such polypeptides, for in vitro purposes related to scientific research, synthesis of DNA and manufacture of DNA vectors.